Hymenolepis diminuta Infection Affects Apoptosis in the Small and Large Intestine

The rat tapeworm Hymenolepis diminuta has been shown to cause alterations in gastrointestinal tissues. Since hymenolepiasis induces a number of reactions in the host, it is reasonable to assume that it may also be involved in the mechanisms of apoptosis in the intestines. Individual research tasks included an examination of the effect of H. diminuta infection on; (i) the cellular localization of the expression of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2, as well as caspase-3 and caspase-9, and (ii) the effects of the infection on the expression of Bcl-2, Bax, Cas-3 and Cas-9, at the mRNA and protein levels. Molecular tests (including mRNA (qRT PCR) and the protein (Western blot) expression of Bax, Bcl-2, and caspases-3, -9) and immunohistochemical tests were performed during the experiment. They showed that H. diminuta infection activates the intrinsic apoptosis pathway in the small and large intestine of the host. H. diminuta infection triggered the apoptosis via the activation of the caspase cascade, including Cas-3 and Cas-9. Hymenolepiasis enhanced apoptosis in the small and large intestine of the host by increasing the expression of the pro-apoptotic gene and protein Bax and by decreasing the expression of the anti-apoptotic gene and protein Bcl-2.


Introduction
Hymenolepis diminuta (rat tapeworm) is a widespread parasite among small rodents and can also occur in humans [1,2]. Despite the fact that the adult tapeworm does not possess clinging organs (hooks) that could damage host tissues, its metabolites can affect the proper functioning of a host's digestive tract [3]. Due to low pathogenicity and putative immunomodulatory activity, H. diminuta can be considered a potential therapeutic agent for the treatment of autoimmune and inflammatory diseases [4][5][6]. To date, the parasite-host system in hymenolepiasis is not yet fully understood in biochemical, histochemical, and molecular terms. It has been shown that hymenolepiasis can cause changes in gastrointestinal tissues. These alterations are mainly related to a decrease in the length of intestinal villi and the deepening of crypts in the digestive system of wild and laboratory rats [7,8].
Metabolites secreted by H. diminuta disrupt the host gastrointestinal function, causing increased saliva secretion, inhibition of gastric juice secretion, and increased trypsin activity in duodenal contents [3]. H. diminuta infection also causes a decrease in transepithelial electrical potential difference, simultaneously causing a blockade of chloride and potassium ion transport in the rat ileum [9,10]. The presence of H. diminuta may also affect smooth muscle contraction in the intestine through the modulation of the muscarinic (M) receptors located in the cell membrane [11] and changes in the composition of intestinal In the planned study, tissues (small and large intestines) from an experiment conducted in 2012-2013 were used, to minimize suffering and the number of animals used. The study was conducted on 30 randomly selected Wistar-strain rats (male) aged approximately 4 months at the start of the experiment.
The rats (n = 30) were divided into five groups: The H. diminuta cysticercoids used to infect the animals had been obtained from cultures from a tapeworm-infected Tribolium destructor dark flour beetle (WMS il 1). The cysticercoids dissected from the body cavity of the insects were washed with saline solution and administered to the rats with an intraesophageal probe (5 cysticercoids in 1 mL of saline).
The experiment was always performed at the same time of day, around 9 a.m. The rats used in the experiment received standard food and water ad libitum. They were kept in a lit room for 12 h per day. A coproscopic examination of the rats' feces for parasites was performed before each experiment (3 weeks after infection).
Rats from both the control and experimental groups (groups I-IV) were sacrificed with Tiopentanal (Biochemie GmbH, Viena, Austria) at a dose of 100 mg/kg body weight (b.w.) intraperitoneally (i.p.), then weighed, and blood and organs collected. The small and large intestine sections collected from the animals were cleaned of adhering connective tissue, dissected along the intestinal mesentery, cleared of digestive contents, and divided into pieces approximately 2 cm long. This biological material was frozen at −80 • C.

An Assay Using MicroBCA Protein Assay Kit and Plate Reader
The collected rat intestinal sections (small intestine and large intestine) were prehomogenized by hammering in liquid nitrogen. RIPA lysis buffer (pH 7.4) contained 20 mM Tris; 0.25 mM NaCl; 11 mM EDTA; 0.5% NP-40, 50 mM sodium fluoride, and protease and phosphatase inhibitors (Roche Diagnostics Deutschland GmbH, Mannheim Germany). The whole mixture was incubated for 2 h at 4 • C with constant shaking. After this time, the samples were centrifuged for 20 min at 15,000 g (at 4 • C); 30 µL was collected for protein determination, and the resulting supernatant was frozen at −80 • C for further analyses.
For the evaluation of Bcl-2, Bax, Cas-3, and Cas-9 protein expression with the Western blot method, the total protein concentration in the test samples was determined using a MicroBCAPierce™ kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions, and a plate reader (BiochromAsys UVM 340). This is a kit that uses a colorimetric method to quantitatively measure total protein in a test sample, compared to a protein standard. Using bicinchoninic acid (BCA), a compound that detects Cu + ions that are formed when a Cu 2+ ion is reduced by a protein in an alkaline medium, a purple color is produced. This is the result of the chelation of two BCA molecules with one copper Cu + ion. The colored water-soluble complex has strong absorbance at 562 nm, which also corresponds to the amount of protein in the test sample.

Analysis of Gene Expression in the Small and Large Intestine by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The quantitative mRNA expression of Bcl-2, Bax, Cas-3, and Cas-9 genes was determined by two-step reverse transcription PCR. The relative expression of the studied genes was determined relative to the average expression of GAPDH, a constitutive expression ref-erence gene (housekeeping genes). Total RNA was isolated from the rat intestinal sections (small and large intestine) using RNeasyMiniKit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The concentration and purity of the isolated RNA were determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific™, Waltham, MA, USA). For cDNA synthesis, 1 µg of total RNA isolated from the tissues was used in a 20 µg total sample volume. The resulting template was transcribed into cDNA using a high-capacity reverse transcription kit with Omniscript RT universal primers (random decamers) (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The quantification of real-time mRNA levels was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with Power SYBR Green PCR Master Mix reagent (Applied Biosystems, Foster City, CA, USA).
The profile of the reaction carried out was as follows: 95 • C (15 s), 40 cycles at 95 • C (15 s), and 60 • C (1 min). The primer sequences were designed to span an intron between a sense primer and an antisense primer to prevent the multiplication of genomic DNA. Each sample was analyzed in duplicate (in two technical replicates), and mean Ct values were used in further studies. An analysis of these relative changes in gene expression between samples was performed using the 2 −∆∆CT method [35] Each reaction ended with a melt curve analysis. The following primer pairs were used (Table 1): Protein electrophoresis was performed using 11% polyacrylamide gel electrophoresis (SDS-PAGE), with 30 µg protein/well for separation. A wet transfer method was then applied, i.e., fractionated proteins were transferred onto a 0.2 µm PVDF membrane (Thermo Fisher Scientific™, Waltham, MA, USA) at 75 V for 60 min. Subsequently, the membrane was blocked with 3% bovine serum albumin (BSA) in blocking buffer for 1 h at room temperature. The expression of apoptotic proteins was determined by immunodetection with specific antibodies. Accordingly, I-primary monoclonal antibodies against Bcl-2 (cat. no: sc-23960, Santa Cruz Biotechnology, Dallas, TX, USA), Cas-3 (cat. no: sc-56053, Santa Cruz Biotechnology, Dallas, TX, USA), Cas-9 (cat.: sc-56076, Santa Cruz Biotechnology, Dallas, TX, USA) and polyclonal against Bax (cat. no: sc-7480, Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:400 overnight at refrigerator temperature. Then, it was incubated with a secondary antimouse antibody at a dilution of 1:4000 for one hour at room temperature. Beta actin (Cat. No: sc-47778, Santa Cruz Biotechnology, USA) was used as the reference protein. To visualize protein expression, an ECL Advance Western Blotting Detection Kit (GE Healthcare, Chicago, IL, USA) was used for chemiluminescence visualization, and the bands were developed using MolecularImagerChemiDock XRS+ (Bio-Rad, Hercules, CA, USA).
2.5. Immunohistochemical Analysis of Bax, BCl-2, Cas-3, and Cas-9 in the Small and Large Intestine Paraffin-embedded sections (3-5 µm) of the small and large intestines of the control and H. diminuta-infected rats were subjected to immunohistochemical staining to visualize the apoptotic proteins Bcl-2, Bax, Cas-3, and Cas-9.
The tissues were placed in a 4% buffered solution of formalin for 24 h, then a dehydration series was performed, and the tissues were embedded in paraffin for 3 h. The blocks were sliced into 3-5 µm thick sections using a microtome (HM 340E Electronic Rotary Microtome, Thermo Fisher Scientific™, Waltham, MA, USA). The sections were then spotted onto histology slides ((3-Aminopropyl)triethoxysilane, Cat. No. J2800AMNZ, Thermo Fisher Scientific™, Waltham, MA, USA), which were deparaffinized and rehydrated. To reveal antigenic determinants, deparaffinization was performed by microwave cooking twice (700 W, 4 and 3 min) in 10 nM citrate buffer (pH 6.0). Immunohistochemistry was performed using overnight incubation at 4 • C with specific primary monoclonal antibodies against Bcl-2 (sc-23960, Santa Cruz Biotechnology, Dallas, TX, USA), Cas-3 (sc-56053, Santa Cruz Biotechnology, Dallas, TX, USA), Cas-9 (cat. no.: sc-56076, Santa Cruz Biotechnology, Dallas, TX, USA), and polyclonal against Bax (cat.: sc-7480, Santa Cruz Biotechnology, Dallas, TX, USA) at a dilution of 1:100. The sections were then stained with an avidinbiotin-peroxidase system with diaminobenzidine as the chromogen. For this purpose, a commercial Dako LSAB + System-HRP kit (Dako Inc., Canpinteria, CA, USA) was used according to the manufacturer's instructions. Brown staining was considered a positive reaction indicating the presence of the antigen, and negative control was performed without primary antibodies. Additionally, hematoxylin contrast staining was used to visualize the cytoplasm and cellular nuclei. A light microscope (Leica DM5000 B, Wetzlar, Germany) integrated with a camera was used to visualize the slides.

Statistical Analysis
Analysis of the obtained results was performed using Statistica 13.0 software (StstSoft Polska sp. z o.o., Kraków, Poland). The arithmetic mean ± SD was calculated for each of the studied parameters. The distribution of results for each variable was obtained using a Shapiro-Wilk test. As most of the obtained results differed from a normal distribution, in further analyses, a non-parametric Kruskal-Wallis test was used to indicate differences between the study groups. Probabilities at p ≤ 0.05 were considered statistically significant.

Changes in Apoptosis Gene Expression in the Small and Large Intestines of the Rat during H. diminuta Infection
The expression of Bcl-2, Bax, Cas-3, and Cas-9 genes in the small and large intestine of the control and H. diminuta infected rats was determined at the mRNA level. The expression of Bax, Cas-3, and Cas-9 genes was lower in the uninfected rats compared to the infected rats. In contrast, an elevated Bcl-2 gene expression in the control group was observed in both the small and large intestines compared to the other groups ( Figure 1).

Changes in Apoptotic Protein Expression in the Small and Large Intestine of the Rat during H. diminuta Infection
H. diminuta infection affected the expression of apoptotic proteins in both the small and large intestine. The expression of Bax protein in the intestines of the infected rats was significantly higher than in the control group. In the small intestine, Bax protein expression increased the most at 8 dpi (122%) (p < 0.01) and less on the other days: 25 dpi (50%) (p < 0.01), 40 dpi (13%), and 60 dpi (80%) (p < 0.01) compared to 0 dpi. In the colon, there was an increase of Bax expression at 8 dpi (28%) (not significant). At 25 dpi (53%), 40 dpi (70%), and 60 dpi (77%) the level of Bax protein decreased significantly (p < 0.05) compared to the 0 dpi group (Figure 3).

Changes in Apoptotic Protein Expression in the Small and Large Intestine of the Rat during H. diminuta Infection
H. diminuta infection affected the expression of apoptotic proteins in both the small and large intestine. The expression of Bax protein in the intestines of the infected rats was significantly higher than in the control group. In the small intestine, Bax protein expression increased the most at 8 dpi (122%) (p < 0.01) and less on the other days: 25 dpi (50%) (p < 0.01), 40 dpi (13%), and 60 dpi (80%) (p < 0.01) compared to 0 dpi. In the colon, there was an increase of Bax expression at 8 dpi (28%) (not significant). At 25 dpi (53%), 40 dpi (70%), and 60 dpi (77%) the level of Bax protein decreased significantly (p < 0.05) compared to the 0 dpi group (Figure 3). The expression of anti-apoptotic protein Bcl-2 in the small intestine varied according to the duration of hymenolepiasis. Protein expression decreased significantly (p < 0.05) by 32% at 8 dpi, and 70% at 25 dpi. On subsequent days, expression was higher vs. the control. At 40 dpi, expression increased by 13% (not significant) and at 60 dpi by 44% (p < 0.05). In the colon of the infected rats, Bcl-2 protein expression was significantly higher at 8 dpi (41%), 25 dpi (43%), and 60 dpi (50%) compared to the uninfected rats (p < 0.01) ( Figure 4).  The expression of anti-apoptotic protein Bcl-2 in the small intestine varied according to the duration of hymenolepiasis. Protein expression decreased significantly (p < 0.05), by 32% at 8 dpi, and 70% at 25 dpi. On subsequent days, expression was higher vs. the control. At 40 dpi, expression increased by 13% (not significant) and at 60 dpi by 44% (p < 0.05). In the colon of the infected rats, Bcl-2 protein expression was significantly higher at 8 dpi (41%), 25 dpi (43%), and 60 dpi (50%) compared to the uninfected rats (p < 0.01) (Figure 4).

H. diminuta Infection
H. diminuta infection affected the expression of apoptotic proteins in both the smal and large intestine. The expression of Bax protein in the intestines of the infected rats was significantly higher than in the control group. In the small intestine, Bax protein expression increased the most at 8 dpi (122%) (p < 0.01) and less on the other days: 25 dpi (50%) (p < 0.01), 40 dpi (13%), and 60 dpi (80%) (p < 0.01) compared to 0 dpi. In the colon, there was an increase of Bax expression at 8 dpi (28%) (not significant). At 25 dpi (53%), 40 dp (70%), and 60 dpi (77%) the level of Bax protein decreased significantly (p < 0.05) compared to the 0 dpi group (Figure 3). The expression of anti-apoptotic protein Bcl-2 in the small intestine varied according to the duration of hymenolepiasis. Protein expression decreased significantly (p < 0.05) by 32% at 8 dpi, and 70% at 25 dpi. On subsequent days, expression was higher vs. the control. At 40 dpi, expression increased by 13% (not significant) and at 60 dpi by 44% (p < 0.05). In the colon of the infected rats, Bcl-2 protein expression was significantly higher at 8 dpi (41%), 25 dpi (43%), and 60 dpi (50%) compared to the uninfected rats (p < 0.01) ( Figure 4).  In the small intestine, caspase-3 protein expression was the highest at 40 dpi, and it was 20% higher compared to the control group (p < 0.05). On subsequent days, expression was lower vs. the control. In the colon, there was a decrease of caspase-3 expression at 8 dpi (43%) (not significant). At 25 dpi, expression decreased by 41%, and at 60 dpi, it decreased by 66% (p < 0.05). In contrast, in the colon, caspase-3 expression was the highest in the uninfected rats, and at 8 (66%) and 25 (73%) days post-infection, the lowest expression of caspase-3 was observed (p < 0.05), increasing at 40 (64%) and 60 (56%) days, but was still significantly lower in the uninfected rat group (p < 0.05) ( Figure 5).
represent the arithmetic mean ± SD and are representative of individual groups of six animals in each experiment. Statistical analysis was performed using the Kruskal-Wallis test, * p < 0.05, vs. 0 dpi.
In the small intestine, caspase-3 protein expression was the highest at 40 dpi, and it was 20% higher compared to the control group (p < 0.05). On subsequent days, expression was lower vs. the control. In the colon, there was a decrease of caspase-3 expression at 8 dpi (43%) (not significant). At 25 dpi, expression decreased by 41%, and at 60 dpi, it decreased by 66% (p < 0.05). In contrast, in the colon, caspase-3 expression was the highest in the uninfected rats, and at 8 (66%) and 25 (73%) days post-infection, the lowest expression of caspase-3 was observed (p < 0.05), increasing at 40 (64%) and 60 (56%) days, but was still significantly lower in the uninfected rat group (p < 0.05) ( Figure 5). For caspase-9 expression in the small intestine, the highest level was observed at 60 dpi (p < 0.05) (148%). From 8 dpi, expression was lower vs. the control. At 8 dpi, expression decreased 17%, at 25 dpi 26%, and 40 dpi 22% (not significant). Caspase-9 expression in the colon at 8 dpi was lower (42%) compared to the control group; however, the change was not significant. The level of Cas-9 protein in the colon significantly increased at 25 dpi (139%) (p < 0.05) vs. the control, and, in subsequent groups, it decreased, reaching a significantly lower level at 60 dpi (66%). At 40 dpi, expression increased by 2% (not significant) ( Figure 6). For caspase-9 expression in the small intestine, the highest level was observed at 60 dpi (p < 0.05) (148%). From 8 dpi, expression was lower vs. the control. At 8 dpi, expression decreased 17%, at 25 dpi 26%, and 40 dpi 22% (not significant). Caspase-9 expression in the colon at 8 dpi was lower (42%) compared to the control group; however, the change was not significant. The level of Cas-9 protein in the colon significantly increased at 25 dpi (139%) (p < 0.05) vs. the control, and, in subsequent groups, it decreased, reaching a significantly lower level at 60 dpi (66%). At 40 dpi, expression increased by 2% (not significant) ( Figure 6). The results of immunohistochemical reactions showed that H. diminuta-infected rats (at 8, 25, 40, and 60 dpi) showed increased Bax, Cas-3, and Cas-9 immunoexpression and

Marker
Part of Intestine  Figure 7 shows Bax immunoexpression in the small intestine of the control animals (0 dpi) and on consecutive days (8 dpi, 25 dpi, 40 dpi, 60 dpi) in the H. diminuta-infected animals. In the small intestinal wall of the control animals (0 dpi) ( Figure 7A,a), the IHC reaction was lowest and was marked in single epithelial cells of the villi and intestinal crypts (red arrows) and in a few cells in the connective tissue lining of the intestinal wall mucosa (blue arrows). By far the strongest immunohistochemical reaction showing Bax expression ( Figure 7B,b) was observed in epithelial cells (red arrows), and the number of immunopositive cells was higher than in control rats (8 dpi). Bax-positive cells were also present in the lamina propria of the mucosa of these animals (blue arrows). On subsequent days after H. diminuta infection, the pattern of Bax expression was similar, but the intensity became lower and lower ( Figure 7C,c,D,d; red and blue arrows) before elevating again by 60 dpi (Figure 7E,e; red and blue arrows). In animals of the 25 dpi group, a few Bax-positive intraepithelial lymphocytes were observed in the epithelium ( Figure 7C; green arrows).
In contrast, in the intestinal wall of the control animals, Bax immunoexpression was very low, and only a few cells of the lining epithelium, mainly cup cells, were Bax-positive ( Figure 8A,a; red arrows). A similar level of IHC reaction was observed in the 40 dpi group ( Figure 8D,d; red arrows) but not in the intestinal crypts. In contrast, at 8, 25, and 60 dpi, the reaction was higher and present mainly in enterocytes ( Figure 8B,b,C,c; red arrows) and cup cells (Figure 8E,e; red arrows). In the lamina propria of the mucosa, Bax-positive cells were present during the early days of hymenolepiasis ( Figure 8B,b,C,c; blue arrows).
The immunoexpression of Bcl-2 in the intestinal wall of animals of all of the infected groups was quite low, while in the control group ( Figure 9A,a) and at 8 dpi ( Figure 9B,b), they were quite comparable, mildly marked in epithelial cells (red arrows), and somewhat stronger in cells of interstitial tissue (blue arrows). At 25 dpi ( Figure 9C,c), the IHC reaction was very weak and barely marked in epithelial cells (red arrows) was and slightly stronger in cells of the mucosa lamina propria (blue arrows). By 40 dpi and 60 dpi, the level of Bcl-2 immunoexpression had increased, and these groups ( Figure 9D  In contrast, in the intestinal wall of the control animals, Bax immunoexp very low, and only a few cells of the lining epithelium, mainly cup cells, were ( Figure 8A,a; red arrows). A similar level of IHC reaction was observed in the ( Figure 8D,d; red arrows) but not in the intestinal crypts. In contrast, at 8, 25 the reaction was higher and present mainly in enterocytes ( Figure 8B,b,C,c;  The immunoexpression of Bcl-2 in the intestinal wall of animals of all of groups was quite low, while in the control group ( Figure 9A,a) and at 8 dpi ( they were quite comparable, mildly marked in epithelial cells (red arrows), an stronger in cells of interstitial tissue (blue arrows). At 25 dpi ( Figure 9C,c), t tion was very weak and barely marked in epithelial cells (red arrows) was  The highest Bcl-2 immunoexpression was observed in the large intestin trol animals ( Figure 10A,a), primarily in the epithelial (cup) cells of intestina arrows), whereas in the lamina propria of the mucosa, Bcl-2-positive cells (b were scarce. At 8 dpi, 25 dpi, 40 dpi, and 60 dpi, the level of Bcl-2 immunoexp comparable to 0 dpi ( Figure 10B,b,C,c,D,d,E,e) and was mainly seen in the gl The highest Bcl-2 immunoexpression was observed in the large intestine of the control animals ( Figure 10A,a), primarily in the epithelial (cup) cells of intestinal crypts (red arrows), whereas in the lamina propria of the mucosa, Bcl-2-positive cells (blue arrows) were scarce. At 8 dpi, 25 dpi, 40 dpi, and 60 dpi, the level of Bcl-2 immunoexpression was comparable to 0 dpi ( Figure 10B,b,C,c,D,d,E,e) and was mainly seen in the glandular epithelium of the intestinal crypts (red arrows). In the control group ( Figure 11A,a), the level of caspase-3 immunoexpression small intestine was lowest, noted in the cytoplasm and nucleus of some epithelia mainly intestinal villi (red arrows). Furthermore, a few cells of the lamina propria submucosa were immunopositive (blue arrows). From 8 dpi with H. diminuta to ( Figure 11B,b,C,c,D,d), the level of caspase-3 immunoexpression gradually increase In the control group ( Figure 11A,a), the level of caspase-3 immunoexpression in the small intestine was lowest, noted in the cytoplasm and nucleus of some epithelial cells, mainly intestinal villi (red arrows). Furthermore, a few cells of the lamina propria of the submucosa were immunopositive (blue arrows). From 8 dpi with H. diminuta to 40 dpi ( Figure 11B,b,C,c,D,d), the level of caspase-3 immunoexpression gradually increased and was characterized by similar localization, that is, in epithelial and interstitial cells (red and blue arrows, respectively), except that the latter were very abundant in the mucosa of animals of the 8 dpi group (Figure 11B,b, blue arrows). The level of caspase-3 immunoexpression in the small intestinal epithelium of the 60 dpi group rats ( Figure 11E,e) was similar to that of the 8 dpi group. In the control animals ( Figure 12A,a), the level of caspase-3 immunoexpression in colon was relatively high, noticeably in epithelial cells (red arrows) and connective ti cells of the mucosal lamina propria (blue arrows). On successive days after H. dimi In the control animals ( Figure 12A,a), the level of caspase-3 immunoexpression in the colon was relatively high, noticeably in epithelial cells (red arrows) and connective tissue cells of the mucosal lamina propria (blue arrows). On successive days after H. diminuta infection (8 dpi, 25 dpi, 40 dpi, 60 dpi), the level of caspase-3 immunoexpression fluctuated: it increased by 8 dpi (Figure 12B,b), decreased by 25 dpi (Figure 12C,c), increased by 40 dpi (Figure 12D,d), and again decreased by 60 dpi (Figure 12E,e). The expression pattern of caspase-3 in the colon wall was similar to the control animals, i.e., it was seen in the epithelium and the mucosal connective tissue (red, blue arrows). In the small intestine of the control animals ( Figure 13A,a), the level of caspase-9 i munoexpression was low and seen in the cytoplasm or nucleus of the epithelial cells (r arrows) and the mucosal lamina propria cells (blue arrows). By 8 dpi ( Figure 13B,b), t In the small intestine of the control animals ( Figure 13A,a), the level of caspase-9 immunoexpression was low and seen in the cytoplasm or nucleus of the epithelial cells (red arrows) and the mucosal lamina propria cells (blue arrows). By 8 dpi ( Figure 13B,b), the level of caspase-9 immunoexpression increased and was seen in the cytoplasm of most epithelial cells (red arrows) and in the interstitial cells (blue arrows). By 25 dpi and 40 dpi, the intensity of immunoreactivity for caspase-9 gradually decreased, with the localization remaining constant ( Figure 13C,c,D,d, red, blue arrows). However by 60 dpi (Figure 13E,e, red, blue arrows), the immunoexpression was comparable to the control group ( Figure 13A,a). In the control animals (0 dpi), caspase-9 immunoexpression in the colon was at a ve low level, with few cells of the epithelium and interstitial tissue being positive for t marker of apoptosis ( Figure 14A,a, red, blue arrows). As time passed after H. dimin In the control animals (0 dpi), caspase-9 immunoexpression in the colon was at a very low level, with few cells of the epithelium and interstitial tissue being positive for this marker of apoptosis ( Figure 14A,a, red, blue arrows). As time passed after H. diminuta infection ( Figure 14B,b,C,c,d,d,E,e), the level of caspase-9 immunoexpression increased linearly. More and more epithelial cells (surface and intestinal crypts) and connective tissues showed cytoplasmic and nuclear immunohistochemical reactions (red, blue arrows).

Discussion
The presence of H. diminuta in the intestine of the host affects the function of th gan and its immunity. Numerous studies indicate the interference of H. diminuta wit host cell response, with cellular signals, and with the invasion mechanism itself. In

Discussion
The presence of H. diminuta in the intestine of the host affects the function of the organ and its immunity. Numerous studies indicate the interference of H. diminuta with the host cell response, with cellular signals, and with the invasion mechanism itself. In our study, we demonstrated the modulation of small and large intestine apoptosis mechanisms during H. diminuta infection by analyzing the expression of specific apoptotic genes and proteins: Bax, Bcl-2, Cas-3, and Cas-9, using quantitative real-time polymerase chain reaction (qRT-PCR), Western blot, and immunohistochemistry (IHC).
It is interesting to note that many parasites have developed a mechanism to modulate host cell apoptosis, thus creating the possibility of easier penetration into the intestinal epithelium. A recent study showed that Giardia sp. can induce apoptosis in intestinal epithelial cells by activating both the intrinsic and extrinsic pathways [36]. Numerous reports suggest that, depending on the strain, this parasite can destructively affect the host intestinal epithelium by inducing different mechanisms. Similar observations have been made for H. diminuta, which causes histopathological changes in the intestines, as well as altering molecular and immunological mechanisms [37].
The present study demonstrates the effect of H. diminuta infection on the apoptosis of host intestinal epithelial cells in a rat model. Molecular and histological studies were performed to determine the induction of apoptotic mechanisms in the small and large intestine and indicated that H. diminuta can stimulate enterocyte apoptosis through the activation of caspase-3 and caspase-9; changes in protein expression, including the high expression of pro-apoptotic proteins (Bax); and the low expression of anti-apoptotic proteins (Bcl-2).
The Bcl-2 family proteins regulate the intrinsic apoptosis pathway centered in mitochondria. The ratios of pro-apoptotic Bax and anti-apoptotic Bcl-2 are correlated with the release of cytochrome C and the subsequent activation of the caspase cascade [38]. Literature data indicates that caspase-3 is the main effector involved in apoptosis, and its activation directs the irreversible step of apoptosis. The release of cytochrome c in mitochondria is one of the initial diagnostic features of nuclear cell apoptosis [39]. Bcl-2 has an anti-apoptotic effect by reducing the release of cytochrome c from mitochondria to inhibit the activation of caspase-3. In contrast, Bax protein is a key component of ion channels located in the mitochondrial membrane. Moreover, it induces cytochrome c translocation across mitochondrial membranes and the formation of apoptotic bodies. Bax activates caspase-9 and caspase-3, which consequently leads to programmed cell death [40]. Therefore, in the present study, the molecular mechanisms associated with such effects are investigated by analyzing the stimulation of hymenolepiasis on the expression levels of cleaved (activated) caspase-3 and caspase-9 and mitochondria-associated apoptotic proteins, Bcl-2 and Bax. In the present study, their expression was checked at both the mRNA and protein levels.
The hypothesis of apoptosis induction by H. diminuta under experimental conditions was confirmed by an observed increase in the expression of the pro-apoptotic protein Bax. Moreover, the theory was supported by immunohistochemical results in which its localization was determined. The study indicates an increased presence of Bax in epithelial cells, lamina propria of the small intestinal mucosa, and enterocytes of cup cells and lamina propria of the colonic mucosa. In addition, Bax-positive intraepithelial lymphocytes were found in the small intestine during hymenolepiasis. In contrast, Bcl-2 expression was significantly lower in the different H. diminuta-infected groups at both the mRNA and protein levels. Similar results were obtained for immunohistochemical analysis. Moreover, the increase in Bax protein shifted the Bax/Bcl-2 ratio in favor of apoptosis. Additionally, it was shown that caspase-3 and caspase-9 were noticeably activated. Caspase-3 and caspase-9 mRNA and protein expressions were significantly higher in the small and large intestine during H. diminuta infection compared to the uninfected control group. The IHC results were analogous to the Western blot and qRT-PCR results and suggest that H. diminuta-induced cell apoptosis was associated with the activation of a mitochondriarelated pathway in the rat intestine.
Similar results apply to infections with other intestinal parasites. Numerous studies show that Giardia sp. affects the host similarly to H. diminuta, except that it also inactivates the PARP protein [32,[41][42][43]. Chin et al. (2002) found that G. lamblia trophozoites can activate the process of apoptosis in non-transformed human small intestinal epithelial cells, depending on the parasite strain. Furthermore, G. lamblia, by inducing apoptosis, reduced the integrity of the host intestinal epithelial barrier. In addition, it was shown that these changes may be dependent on caspase-3 [41], whose increased activity was also observed during H. diminuta infection. Panaro et al. (2007) conducted a study on apoptosis in a parasite-host system using human ileal adenocarcinoma line cells (HCT-8). The interaction of colon epithelial cells with G. intestinalis trophozoites showed that the parasite can modulate apoptosis processes in the host intestines through DNA fragmentation, caspase-3 stimulation, PARP degradation, and the regulation of protein expression: Bcl-2 and Bax [36]. An experiment by Troeger et al. (2007) showed only a slight effect of G. lamblia on the apoptosis process among patients infected with G. lamblia, where it increased by 0.2% compared to the control group [43]. In contrast, Roxstrom-Lindquist et al. (2005) suggest that this parasite not only can induce apoptosis but also induce large changes in cell gene expression, including pro-inflammatory factors in intestinal epithelial cells [42]. These reports indicate that the adaptive mechanisms involving the production of surface proteins and the secretion of secretory products may be responsible for the induction of apoptosis glideos [37].
Analyzing the results obtained, it can be assumed that both the mere presence of the parasite in the intestines and its ES may indicate the pro-apoptotic potential of H. diminuta and the putative function of caspase-dependent apoptosis in the pathogenesis of hymenolepiasis. Given the location of the H. diminuta and its impact on normal intestinal function, studies of the modulation of apoptotic pathways in the intestines in the parasitehost system are still a point of interest. H. diminuta is known to induce mucosal barrier changes during infection [7,10].
A recent study by Hayes et al. (2017) demonstrates that cell proliferation and apoptosis both increase in the colon during chronic infection in mice with Trichuris muris. A significant increase in the number of cells indicative of apoptosis was observed at the base of the crypts, while in more infected animals, successively more proliferating cells were found further down the intestinal crypt. Interestingly, no effect of infection on proliferation or apoptosis was observed in the small intestine, despite the presence of morphological changes at this site [44]. In contrast, in the present study, the immunoexpression of the pro-apoptotic protein Bax and caspase-3 and caspase-9 were significantly higher in both the small intestine (the site of the parasite) and the large intestine, indicating increased cell turnover in these structures. Although the target site of H. diminuta is the small intestine, the observed changes in the large intestine may suggest the secretion of substances into the intestinal lumen, simultaneously inducing apoptotic processes in later parts of the gastrointestinal tract.
Target cell death is associated with the contact-dependent caspase activation of infected cells [45]. In the present study, the expression of caspase-3, as well as caspase-9, was elevated in both mRNA and protein levels compared to the control group. Thus, the extracellular parasite can induce apoptosis in cells independently of the membrane death receptors, i.e., the external apoptotic pathway and of the internal apoptotic pathway. Thus, it can induce apoptosis through direct contact upon attachment to the intestinal epithelium. Similar observations apply to Entamoeba histolytica infections. The cytotoxicity of E. histolytica has also been shown to depend on effector proteins, which include amoebapore and proteases responsible for parasite invasion and host cell apoptosis. In addition, these proteins are thought to be responsible for the direct activation of caspase-3 [46].
In hymenolepiasis, changes in intestinal morphology are observed, including atrophy of intestinal villi [8]. Similar observations have been noted in Nippostrongylus brasiliensis infection. A study by Hyoh et al. (1999) showed that this parasite can cause partial villous atrophy and crypt proliferation in the small intestinal mucosa. Furthermore, it was shown that intestinal villi atrophy was associated with increased apoptosis and a loss of adhesion in host epithelial cells [47]. Hence, it can be speculated that H. diminuta infection inducing analogously similar changes in intestinal structures may induce the same apoptotic mechanism as N. brasiliensis. Thus, the induction of the apoptosis pathway in intestinal epithelial cells may be linked to the host defense mechanism of rapidly removing damaged cells. It is supposed that the apoptosis of villi cells can be induced directly by molecules from the parasite itself, as well as from ES [48,49].
In an experiment by Kuroda et al. (2002), the effects of N. brasiliensis and ES on the intestinal epithelial cell line IEC-6 were examined. The study indicates that N. brasiliensis secretes biologically active molecules that can induce apoptosis in host intestinal epithelial cells along with the upregulation of Fas expression. Although the mechanism of apoptosis induction itself remains undocumented, exposure to ES has been shown to trigger apoptosis mechanisms in cells, including nuclear fragmentation, caspase-3 activation, and specific PARP cleavage [50].
H. diminuta, similarly to the other mentioned intestinal parasites, may contribute not only to single-cell disorders but also to the functioning of the whole organ, especially as the parasites have evolved complex mechanisms to modulate parasite-host interactions. It has been observed that Cryptosporidium, depending on the species and the particular stages of the developmental cycle, also affects the mechanisms of host apoptosis pathways [51][52][53][54][55][56][57].
Our study demonstrated that H. diminuta can induce apoptosis in the host intestines. A significant increase in the expression of caspase-3 and caspase-9, which is correlated with the activation of the caspase cascade during apoptosis, indicates that H. diminuta induces the intrinsic pathway of apoptosis at the parasite target site [34,35]. Our data show that apoptosis is only one of many mechanisms occurring during intestinal parasite infections and that the parameters determined in this study may be useful markers of apoptosis in parasitic diseases. Many questions remain unanswered, such as whether increased induction of physiological turnover of intestinal epithelial cells is stimulated at the immune level to remove parasites by causing their eventual expulsion along with their immediate environment, the intestinal epithelium; how the inhibition of apoptotic pathways affects the host intestinal epithelium; and whether anti-apoptotic treatment during infection with these parasites is possible without harm to other tissues.
The modulation of host apoptosis under parasitic invasion is not yet fully understood and requires further study on other intestinal parasites. It opens an interesting new area of research due to the wide range of subject matter and the increasing number of infections with "new" and opportunistic intestinal parasites. Furthermore, since there is no clear and publicly available animal model for intestinal parasite infections, future experiments should also focus on the development of animal models to support in vitro studies with data obtained in the parasite-host system.

Conclusions
Infection with H. diminuta: (i) activates the intrinsic apoptotic pathway in the small and large intestine of the host; (ii) triggers apoptosis via the activation of a caspase cascade, such as with caspase-3 and caspase-9; (iii) enhances apoptosis in the small and large intestine of the host by increasing the expression of the gene and pro-apoptotic protein Bax and by decreasing the gene and protein expression of anti-apoptotic Bcl-2.
Programmed cell death is an important process for both parasite survival and pathogenicity and for the host to maintain defense mechanisms.  Informed Consent Statement: Informed consent was obtained from all subjects.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.